Non-toxic concentrations of cadmium inhibit systemic movement of turnip vein clearing virus by a salicylic acid-independent mechanism

Systemic movement of plant viruses is a central event in viral infection. To better understand this process, the heavy metal cadmium was used to inhibit systemic spread of turnip vein clearing virus (TVCV), a tobamovirus, in tobacco plants. Study of the mechanism by which cadmium exerts this inhibitory effect may provide insights into the essential steps of the TVCV systemic movement pathway. Our results demonstrated that cadmium treatment did not affect TVCV transport from the inoculated non-vascular tissue into the plant vasculature but blocked viral exit into uninoculated non-vascular tissues. Thus, TVCV virions may enter and exit the host plant vascular system by two different mechanisms. We also showed that cadmium-treated plants still supported systemic spread of an unrelated tobacco etch virus (TEV), suggesting multiple pathways for systemic infection. Finally, cadmium-induced arrest in TVCV systemic infection was shown to occur by a salicylic acid-independent mechanism.     

Movement and subcellular localization of a tobamovirus in Arabidopsis

Tobamoviruses represent a well-characterized system used to examine viral infection, whereas Arabidopsis is a choice plant for most genetic experiments. It would be useful to combine both approaches into one experimental system for virus–plant interaction. Most tobamoviruses, however, are not pathogenic in Arabidopsis . Here, we describe infection of Arabidopsis by a recently discovered crucifer-infecting turnip vein clearing tobamovirus (TVCV). Using this system, we determined patterns and kinetics of viral local and systemic movement within Arabidopsis plants. Localization studies showed that the virus infects both vegetative and reproductive plant tissues. However, there may be a transport barrier between the seed coat and the embryo which virions cannot cross, preventing seed transmission of TVCV. The ability to move both locally and systemically in Arabidopsis , causing mild and fast-developing symptoms but allowing survival and fertility of the infected plants, distinguish TVCV infection of Arabidopsis as a model system to study virus–plant interaction.     

Inhibition of plant viral systemic infection by non‐toxic concentrations of cadmium

Heavy metals, such as cadmium, have a significant impact on plant physiology. However, their potential effect on plant–pathogen interaction, an important biological process, has not been examined. This study shows that exposure of tobacco plants to non-toxic concentrations of cadmium completely blocked viral disease caused by turnip vein clearing virus. Cadmium-mediated viral protection was due to inhibition of the systemic movement of the virus, i.e. its spread from the inoculated into uninoculated leaves. Exposure of plants to cadmium had no effect on viral replication, assembly and local movement within the inoculated leaf. Analysis of the viral presence in different tissues suggested that cadmium treatment inhibited virus exit from the vascular tissue into uninoculated leaves rather than its entry into the host plant vasculature. Higher, toxic levels of cadmium did not produce this inhibitory effect on viral movement, allowing the systemic spread of the virus and development of the viral disease. These observations suggest that cadmium-induced viral protection requires a relatively healthy, unpoisoned plant in which non-toxic levels of cadmium may trigger the production of cellular factors which interfere with the viral systemic movement.     

Transgenic expression of pectin methylesterase inhibitors limits tobamovirus spread in tobacco and Arabidopsis

Plant infection by a virus is a complex process influenced by virus‐encoded factors and host components which support replication and movement. Critical factors for a successful tobamovirus infection are the viral movement protein (MP) and the host pectin methylesterase (PME), an important plant counterpart that cooperates with MP to sustain viral spread. The activity of PME is modulated by endogenous protein inhibitors (pectin methylesterase inhibitors, PMEIs). PMEIs are targeted to the extracellular matrix and typically inhibit plant PMEs by forming a specific and stable stoichiometric 1:1 complex. PMEIs counteract the action of plant PMEs and therefore may affect plant susceptibility to virus. To test this hypothesis, we overexpressed genes encoding two well‐characterized PMEIs in tobacco and Arabidopsis plants. Here, we report that, in tobacco plants constitutively expressing a PMEI from Actinidia chinensis (AcPMEI), systemic movement of Tobacco mosaic virus (TMV) is limited and viral symptoms are reduced. A delayed movement of Turnip vein clearing virus (TVCV) and a reduced susceptibility to the virus were also observed in Arabidopsis plants overexpressing AtPMEI‐2. Our results provide evidence that PMEIs are able to limit tobamovirus movement and to reduce plant susceptibility to the virus.     

Systemic movement of a tobamovirus requires host cell pectin methylesterase

Systemic movement of plant viruses through the host vasculature, one of the central events of the infection process, is essential for maximal viral accumulation and development of disease symptoms. The host plant proteins involved in this transport, however, remain unknown. Here, we examined whether or not pectin methylesterase (PME), one of the few cellular proteins known to be involved in local, cell-to-cell movement of tobacco mosaic virus (TMV), is also required for the systemic spread of viral infection through the plant vascular system. In a reverse genetics approach, PME levels were reduced in tobacco plants using antisense suppression. The resulting PME antisense plants displayed a significant degree of PME suppression in their vascular tissues but retained the wild-type pattern of phloem loading and unloading of a fluorescent solute. Systemic transport of TMV in these plants, however, was substantially delayed as compared to the wild-type tobacco, suggesting a role for PME in TMV systemic infection. Our analysis of virus distribution in the PME antisense plants suggested that TMV systemic movement may be a polar process in which the virions enter and exit the vascular system by two different mechanisms, and it is the viral exit out of the vascular system that involves PME.     

Interaction between the tobacco mosaic virus movement protein and host cell pectin methylesterases is required for viral cell-to-cell movement

Virus-encoded movement protein (MP) mediates cell-to-cell spread of tobacco mosaic virus (TMV) through plant intercellular connections, the plasmodesmata. The molecular pathway by which TMV MP interacts with the host cell is largely unknown. To understand this process better, a cell wall-associated protein that specifically binds the viral MP was purified from tobacco leaf cell walls and identified as pectin methylesterase (PME). In addition to TMV MP, PME is recognized by MPs of turnip vein clearing virus (TVCV) and cauliflower mosaic virus (CaMV). The use of amino acid deletion mutants of TMV MP showed that its domain was necessary and sufficient for association with PME. Deletion of the PME-binding region resulted in inactivation of TMV cell-to-cell movement.     

Chemically induced virus resistance in Arabidopsis thaliana is independent of pathogenesis-related protein expression and the NPR1 gene

Salicylic acid (SA) treatment triggers inhibition of replication or movement of several positive-sense RNA plant viruses in tobacco. This resistance can also be stimulated by nonlethal concentrations of cyanide and antimycin A (AA) without triggering induction of pathogenesis-related PR-1 protein genes. In two ecotypes of Arabidopsis thaliana (Columbia and N?ssen), SA-induced resistance to a tobamovirus, Turnip vein clearing virus (TVCV), was also induced by nonlethal concentrations of cyanide and AA without concomitant induction of PR-1 gene expression. Furthermore, chemically induced resistance to TVCV, as well as the induction of the plant mitochondrial alternative oxidase (a potential target for the chemicals), was independent of NPR1, a gene that plays a key role downstream of SA in the induction of PR proteins. The chemically induced resistance to TVCV appeared to be due to inhibition of replication at the site of inoculation. Taken together, these results show that in Arabidopsis, as in tobacco, resistance to viruses can be induced via a distinct branch of the defensive signal transduction pathway. This suggests that the existence of this virus-specific branch may be widespread among plants.     

Effect of lead (Pb) on the systemic movement of RNA viruses in tobacco (Nicotiana tabacum var. Turkish)

Effect of various lead (Pb) concentrations on the systemic movement of RNA viruses was examined in tobacco plants. Prior to inoculation, plants were grown hydroponically for 6 days in Hoagland’s solution supplemented with five concentrations of lead nitrate [Pb(NO₃)₂]: 0.0 (control), 10, 15, 50, and 100 μM. Four different RNA viruses with different cell-to-cell movement mechanisms were used. Two weeks after inoculation lower and upper leaves of each treatment were harvested and examined for the presence of viral coat protein. In plants inoculated with Tobacco mosaic virus, Potato virus X, and Tobacco etch virus, TEM images and western blot assays confirmed the presence of viral coat proteins in the upper leaves of all lead treatments. However, in plants inoculated with Turnip vein-clearing virus (TVCV), no signs of viral particles were detected in the upper leaves of plants treated with 10 μM or 15 μM lead nitrate. In contrast, plants treated with high concentrations of lead nitrate (50 μM or 100 μM) showed viral particles in their upper leaves. In plants treated with 10 μM or 15 μM lead nitrate, callose accumulation was the same as in control plants. This suggests that non-toxic concentrations of lead nitrate may trigger the production of putative cellular factors in addition to callose that interfere with the TVCV systemic movement. In contrast, plants treated with 100 μM lead nitrate showed less callose as compared to control plants, facilitating the systemic movement of TVCV.     

Turnip vein clearing virus movement protein nuclear activity: Do Tobamovirus movement proteins play a role in immune response suppression?

Plant viruses'' cell-to-cell movement requires the function of virally encoded movement proteins (MPs). The Tobamovirus, Tobacco mosaic virus (TMV) has served as the model virus to study the activities of single MPs. However, since TMV does not infect the model plant Arabidopsis thaliana I have used a related Tobamovirus, Turnip vein-clearing virus (TVCV). I recently showed that, despite belonging to the same genus, the behavior of the 2 viruses MPs differ significantly during infection. Most notably, MP^TVCV, but not MP^TMV, targets the nucleus and induces the formation of F actin-containing filaments that associate with chromatin. Mutational analyses showed that nuclear localization of MP^TVCV was necessary for TVCV local and systemic infection in both Nicotiana benthamiana and Arabidopsis. In this addendum, I propose possible targets for the MP^TVCV nuclear activity, and suggest viewing MPs as viral effector-like proteins, playing a role in the inhibition of plant defense.     

Replication in the phloem is not necessary for efficient vascular transport of tobacco mosaic tobamovirus

Plant viruses move systemically from one leaf to another via phloem. However, the viral functions needed for systemic movement are not fully elucidated. An experimental system was designed to study the effects of low temperature on the vascular transport of the tobacco mosaic tobamovirus (TMV). Vascular transport of TMV from lower inoculated leaves to upper non-inoculated leaves via a stem segment kept at low temperature (4 degrees C) was not affected. On the other hand, several experiments were performed on tobacco leaves to demonstrate that virus replication did not occur at the same temperature. The data suggest that replication of TMV in the phloem of wild-type tobacco plants is not necessary for the vascular transport of TMV, and that the virus moves with photoassimilates as suggested previously.     

The Ins and Outs of Nondestructive Cell-to-Cell and Systemic Movement of Plant Viruses

Propagation of viral infection in host plants comprises two distinct and sequential stages: viral transport from the initially infected cell into adjacent neighboring cells, a process termed local or cell-to-cell movement, and a chain of events collectively referred to as systemic movement that consists of entry into the vascular tissue, systemic distribution with the phloem stream, and unloading of the virus into noninfected tissues. To achieve intercellular transport, viruses exploit plasmodesmata, complex cytoplasmic bridges interconnecting plant cells. Viral transport through plasmodesmata is aided by virus-encoded proteins, the movement proteins (MPs), which function by two distinct mechanisms: MPs either bind viral nucleic acids and mediate passage of the resulting movement complexes (M-complexes) between cells, or MPs become a part of pathogenic tubules that penetrate through host cell walls and serve as conduits for transport of viral particles. In the first mechanism, M-complexes pass into neighboring cells without destroying or irreversibly altering plasmodesmata, whereas in the second mechanism plasmodesmata are replaced or significantly modified by the tubules. Here we summarize the current knowledge on both local and systemic movement of viruses that progress from cell to cell as M-complexes in a nondestructive fashion. For local movement, we focus mainly on movement functions of the 30 K superfamily viruses, which encode MPs with structural homology to the 30 kDa MP of Tobacco mosaic virus, one of the most extensively studied plant viruses, whereas systemic movement is primarily described for two well-characterized model systems, Tobacco mosaic virus and Tobacco etch potyvirus. Because local and systemic movement are intimately linked to the molecular infrastructure of the host cell, special emphasis is placed on host factors and cellular structures involved in viral transport.     

Plant virus transport: motions of functional equivalence

Plant virus cell-to-cell movement and subsequent systemic transport are governed by a series of mechanisms involving various virus and plant factors. Specialized virus encoded movement proteins (MPs) control the cell-to-cell transport of viral nucleoprotein complexes through plasmodesmata. MPs of different viruses have diverse properties and each interacts with specific host factors that also have a range of functions. Most viruses are then transported via the phloem as either nucleoprotein complexes or virions, with contributions from host and virus proteins. Some virus proteins contribute to the establishment and maintenance of systemic infection by inhibiting RNA silencing-mediated degradation of viral RNA. In spite of all the different movement strategies and the viral and host components, there are possible functional commonalities in virus-host interactions that govern viral spread through plants.     

MPB2C, a microtubule-associated plant factor, is required for microtubular accumulation of tobacco mosaic virus movement protein in plants

Movement protein binding 2C (MPB2C) is a plant endogenous microtubule-associated protein previously identified as an interaction partner of tobacco (Nicotiana tabacum) mosaic virus movement protein (TMV-MP). In this work, the role of MPB2C in cell-to-cell transport of TMV-MP, viral spread of TMV, and subcellular localization of TMV-MP was examined. To this end, plants with reduced MPB2C levels were generated by a gene-silencing strategy. Local and systemic spread of TMV and cell-to-cell movement of TMV-MP were unimpaired in MPB2C-silenced plants as compared to nonsilenced plants, indicating that MPB2C is not required for intercellular transport of TMV-MP itself or spread of TMV. However, a clear change in subcellular distribution of TMV-MP characterized by a nearly complete loss of microtubular localization was observed in MPB2C-silenced plants. This result shows that the MPB2C is a central player in determining the complex subcellular localization of TMV-MP, in particular its microtubular accumulation, a phenomenon that has been frequently observed and whose role is still under discussion. Clearly, MPB2C mediated accumulation of TMV-MP at microtubules is not required for intercellular spread but may be a means to withdraw the TMV-MP from the cell-to-cell transport pathway.     

MPB2C,a microtubule-associated plant protein binds to and interferes with cell-to-cell transport of tobacco mosaic virus movement protein

The movement protein of tobacco mosaic virus, MP30, mediates viral cell-to-cell transport via plasmodesmata. The complex MP30 intra- and intercellular distribution pattern includes localization to the endoplasmic reticulum, cytoplasmic bodies, microtubules, and plasmodesmata and likely requires interaction with plant endogenous factors. We have identified and analyzed an MP30-interacting protein, MPB2C, from the host plant Nicotiana tabacum. MPB2C constitutes a previously uncharacterized microtubule-associated protein that binds to and colocalizes with MP30 at microtubules. In vivo studies indicate that MPB2C mediates accumulation of MP30 at microtubules and interferes with MP30 cell-to-cell movement. In contrast, intercellular transport of a functionally enhanced MP30 mutant, which does not accumulate and colocalize with MP30 at microtubules, is not impaired by MPB2C. Together, these data support the concept that MPB2C is not required for MP30 cell-to-cell movement but may act as a negative effector of MP30 cell-to-cell transport activity.     

A novel function for a ubiquitous plant enzyme pectin methylesterase: the host-cell receptor for the tobacco mosaic virus movement protein

Plant virus-encoded movement proteins promote viral spread between plant cells via plasmodesmata. The movement is assumed to require a plasmodesmata targeting signal to interact with still unidentified host factors presumably located on plasmodesmata and cell walls. The present work indicates that a ubiquitous cell wall-associated plant enzyme pectin methylesterase of Nicotiana tabacum L. specifically binds to the movement protein encoded by tobacco mosaic virus. We also show that pectin methylesterase is an RNA binding protein. These data suggest that pectin methylesterase is a host cell receptor involved in cell-to-cell movement of tobacco mosaic virus.     

Effects of Salicylate on Systemic Invasion of Tobacco Plants by Various Viruses

Salicylate watered onto soil in which White Burley tobacco plants were grown represents a reversible stress characterized by stomatal closure, slight slackening of plant growth and low chlorophyll loss. Salicylate affected viral pathogenesis in opposite ways. It had no effect against local and systemic infections by potato virus X (PVX), potato virus Y⁰ (PVY⁰) or tobacco mosaic virus (TMV), whereas it completely prevented systemic infection by alfalfa mosaic virus (AIMV) or tobacco, rattle virus (TRV) in a high proportion of treated plants. When infection moved from leaves inoculated with AIMV or TRV, the tendency to limit systemic spread was shown by the restriction of systemic infection to very limited areas erratically distributed in some uninoculated leaves. The salicylate-induced restriction of AIMV or TRV infectivity to inoculated leaves did not appear due to inhibition of virus multiplication because the inoculation of potentially resistant leaves of salicylate-reated plants resulted in virus antigen accumulation comparable to that of untreated controls. Salicylate may therefore inhibit some long distance virus transport function. Salicylate appears able to evoke true hypersensitivity only against systemic viruses able to induce local necrotic lesions, probably by activating some genetic information for resistance that is normally not expressed.     

Virulence and differential local and systemic spread of cucumber mosaic virus in tobacco are affected by the CMV 2b protein

A mutant of the Cucumber mosaic virus subgroup IA strain Fny (Fny-CMV) lacking the gene encoding the 2b protein (Fny-CMVdelta2b) induced a symptomless systemic infection in tobacco. Both the accumulation of Fny-CMVdelta2b in inoculated tissue and the systemic movement of the virus appeared to proceed more slowly than for wild-type Fny-CMV. The influence of the 2b protein on virus movement in the inoculated leaf was examined using viral constructs derived from Fny-CMV and Fny-CMVdelta2b expressing the green fluorescent protein. Laser scanning confocal microscopy was used to visualize the movement of these viruses. Whereas the wild-type virus spread between the epidermal cells as well as the mesophyll cells, the mutant virus spread less efficiently through the epidermal layer and moved preferentially through the mesophyll. Thus, the 2b protein of Fny-CMV influences the dynamics of movement of the virus both within the inoculated leaf and through the whole plant. We propose that this altered movement profile of Fny-CMVdelta2b results in the absence of disease symptoms in tobacco.     

Down-regulation of the 26S proteasome subunit RPN9 inhibits viral systemic transport and alters plant vascular development

Plant viruses utilize the vascular system for systemic movement. The plant vascular network also transports water, photosynthates, and signaling molecules and is essential for plant growth. However, the molecular mechanisms governing vascular development and patterning are still largely unknown. From viral transport suppressor screening using virus-induced gene silencing, we identified a 26S proteasome subunit, RPN9, which is required for broad-spectrum viral systemic transport. Silencing of RPN9 in Nicotiana benthamiana inhibits systemic spread of two taxonomically distinct viruses, Tobacco mosaic virus and Turnip mosaic virus. The 26S proteasome is a highly conserved eukaryotic protease complex controlling many fundamental biochemical processes, but the functions of many 26S proteasome regulatory subunits, especially in plants, are still poorly understood. We demonstrate that the inhibition of viral systemic transport after RPN9 silencing is largely due to alterations in the vascular tissue. RPN9-silenced plants display extra leaf vein formation with increased xylem and decreased phloem. We further illustrate that RPN9 functions at least in part through regulation of auxin transport and brassinosteroid signaling, two processes that are crucial for vascular formation. We propose that RPN9 regulates vascular formation by targeting a subset of regulatory proteins for degradation. The brassinosteroid-signaling protein BZR1 is one of the targets.